I. SUMMARY OF PARENT GRANT GM126488 The proposed studies of the parental grant are focused on the study of a novel eukaryotic 5 end cap modification. Regulation of RNA stability is an important determinant of the post-transcriptional control of eukaryotic gene expression. Minor alterations in mRNA stability can have profound consequences and may manifest as clinical phenotypes. Eukaryotic mRNAs are generally thought to possess an N7 methyl guanosine (m7G) cap at their 5 end to promote their stability and translation. We recently demonstrate that mammalian RNAs can also be modified to carry a 5'-end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support stability and translation but instead promotes mRNA decay of exogenously transfected RNAs. NAD+ capped RNAs consist of mRNAs and noncoding RNAs, including intronic encoded small noncoding RNAs demonstrating a broad range of RNAs that can be NAD+ capped. Furthermore, we identified the noncanonical DXO decapping enzyme as a protein that efficiently removes the NAD+ cap from the 5 end of RNAs both in vitro and in cells, in a process termed ?deNADding?. We build on these novel findings to characterize the mechanism and function of NAD+ capping and deNADding with three Aims: 1) identify the genome-wide spectrum of NAD+-capped RNAs and assess their function in stability; 2) delineate the NAD+ capping mechanism and 3) begin assess the regulation of deNADding with the identification of NAD+-cap binding proteins. Collectively, we have established NAD+ as an alternative mammalian RNA cap and the proposed studies will provide insight into a heretofore unknown fundamental post-transcriptional regulatory mechanism and will provide the framework for novel avenues to control gene expression in normal and disease states.